Intracellular pH-measurements in rat duodenal mucosa in vitro using confocal laserscan microscopy.

An improved technique was developed to measure intracellular pH-changes in in vitro duodenal mucosa. A confocal laserscan microscope was equipped with a second laser to permit dual wavelength excitation measurements employing BCECF (2'7'-bis-2-carboxyethyl-5-(and-6)carboxyfluorescein), a pH-sensitive fluorescent dye. Intact rat duodenal epithelium was mounted in a microperfusion chamber and loaded with BCECF via submucosal injection. Viability of the epithelial cells could be directly monitored by estimating the nucleus-to-cytoplasm ratio of the fluorescence intensity of BCECF. A calibration procedure using isolated duodenal cells, allowed estimation of the apparent intracellular pH. Initial apparent intracellular pH was 7.32 +/- 0.12, identical to that of isolated duodenocytes. Exposure of the duodenal epithelium to 60 mM NH4Cl led to a steady increase in apparent intracellular pH of 0.46 units within 2 min. Luminal application of 0.01 N HCl led to a steady decrease in apparent intracellular pH of 0.53 pH units within 1 min and was followed by a slow increase to baseline level after acid removal. Thus, confocal laserscan microscopy in combination with BCECF allowed noninvasive monitoring of intracellular pH-changes in single cells of an intact duodenal epithelium.